Method for angiogenesis inhibition or immunostimulation

ABSTRACT

The present invention relates to a composition for use in prevention or treatment of a vascular-related disease, particularly used for angiogenesis inhibition, tumor growth inhibition or tumor metastasis inhibition, or immunostimulation, which comprises glutamic acid or derivatives thereof, preferably glutamic acid is anhydrous glutamic acid represented by Formula (1) or pyroglutamic acid, and a pharmaceutically acceptable carrier or an edible carrier, and a method of preventing or treating a vascular-related disease.

The present application is based on Japanese Patent Application No.2002-177774, Japanese Patent Application filed Apr. 7, 2003, andJapanese Patent Application No. 2003-131997, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for angiogenesis inhibition,neoplasm depression or immunostimulation. etc. which comprisesadministering to a person or an animal anhydrous glutamic acid,pyroglutamic acid, or derivative thereof as active ingredient.

2. Prior Art

A vascular-related disease includes, for example, propagation andtransition of neoplasm, inflammation, rheumatoid arthritis, diabeticretinopathy, and psoriasis, and it is greatly related with angiogenesisand immune function. Angiogenesis is phenomenon of generating newcapillary blood vessels in animal tissues or organs, in the process ofwhich, vascular basement membrane is disassembled and attacked byprotease, vascular endothelial cell is grown by migration and bonded toextra cellular matrix, vascular endothelial cell is differentiated, andvascular cavity is formed. In general, new blood vessels are formed andextended in childhood and growth period. However, when growth period ispast, the occasion of angiogenesis in the body is limited. Angiogenesisis observed under normal physiology condition such as luteinization,ovulation, embryogenesis, and placentation, and also occurred in cureprocess of lesion and resealing process of inflammation. As describedabove, angiogenesis occurs in normal state and has an important role inrestoration of tissues, but it is known that capillary increases in alot of chronic diseases such as diabetes mellitus and that angiogenesiscauses grave lesion to tissues.

Angiogenesis is involved in etiology and aggravation of case of variousdiseases. The diseases include enhancement and transition of malignant,diabetic retinopathy, neovascular glaucoma, inflammatory dermatosis,joint fluid rheumatism, osteoarthritis, atherosclerosis, and obstructiveaffection such as myocardial infarction.

For example, when malignant tumor multiplies, tumor cell inducesvascular neogenesis for itself by angiogenesis promoter to getnourishment and oxygen which are necessary for propagation of tumorcell, and tumor cell further grows while getting nutrient through newblood vessels. Transition of tumor cell to other organ and site alsoinduces angiogenesis, and tumor cell is carried by the bloodstream. Inthe case of diabetic retinopathy, capillary is clogged up because of theviscosity blood set up by diabetes mellitus and is affected, andbleeding and edema are produced in retina. When bleeding and edema arechronic, the retina falls short of oxygen and nourishment, and thennewborn blood vessel originates on the retina or nervous system mammillaso that fiber tissue is formed circumferentially. The retina is pulledby means of the fiber tissue (retinal detachment) or the retinal bloodvessel is occurred bleeding (vitreous hemorrhage), and then seriousvisual handicap or blindness is occurred before long.

As described above, because angiogenesis is deeply involved in the onsetand development of various diseases, a lot of searches of materialsinhibiting angiogenesis have been done hitherto and investigation ispushed forward at the present zealously as an aim in a treatment orprevention of these diseases. As material and drug with actioninhibiting angiogenesis, sulfation polysaccharide (Japanese PatentLaid-Open No. S63-119500 bulletin), trafermin, heparin and steroid (U.S.Pat. No. 4,994,443 specification; or U.S. Pat. No. 5,001,116specification), ascorbic acid ether and this related-compound (JapanesePatent Laid-Open No. S58-131978 bulletin), interferon alpha orinterferon beta (Sidky et al., “Cancer Research”, 47:5155-5161, (1987)),thiazole derivative (Japanese Patent Publication No. H6-62413 bulletin),shark cartilage extract (chondroitin and mucopolysaccharide) (JapanesePatent Laid-Open No. H10-147534 bulletin), polysaccharide derived fromstreptococcus bacteria (Japanese Patent Publication No. H6-62426bulletin), O-displacement fumagillol derivative (Japanese Patent No.3120187 bulletin), neo agarose oligosaccharide (Japanese Patent No.3071068th bulletin) etc. are proposed.

However, considering practical side, all of the materials having actionof angiogenesis inhibition proposed or examined till now as a materialdid not show the sufficiently satisfied effect. The materials were usedbased on experimental findings under the dosage condition that is notpractical, the materials had worries about adverse reactions, or thematerials must be administered in high doses in use. Therefore, thedevelopment of materials by which angiogenesis is inhibited moreeffectively and materials to be used without any worry in a point ofsafety is demanded.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a method forangiogenesis inhibition that inhibits angiogenesis strongly, method forneoplasin inhibition, or method for immunostimulation.

According to the feature of the invention, a method for ‘angiogenesisinhibition’ or ‘immunostimulation’ comprises administering to a personor an animal anhydrous glutamic acid represented by Formula (1),pyroglutamic acid represented by Formula (2), or a salt or amide thereofand a pharmaceutically acceptable carrier or an edible carrier.

In accordance with a preferred embodiment of the invention, theanhydrous glutamic acid and pyroglutamic acid are L-type or DL-type. Theanhydrous glutamic acid, pyroglutamic acid and said salt or amidethereof are used in form of fruit body of Basidiomycetes or mycelium, adry powder thereof, or an extract or a purified material thereof.Preferably, the Basidiomycetes is one or more kinds selected from thegroup consisting of Lentinus edodes, Flammulina velutipes, Lyophyllumaggregatum, Pleurotus ostreatus, Agaricus fungus, Phellinus linteusfungus, Ganoderma lucidum, Hericium Erinaceum fungus, Coriolusversicolor, Agaricus campestris, Grifola frondosa, Sparassis erispa,Schizophyllum commune, Tremella fuciformis Berkeley, and Cordycepssinensis (tochukaso).

The extract is extracted by using “water and/or a hydrophilic organicsolvent”, or “water and/or a hydrophilic organic solvent and ahydrophobic organic solvent”. The hydrophilic organic solvent ismethanol, ethanol, acetone or propanol, and the hydrophobic organicsolvent is hexane or chloroform. The method is used for tumor growthinhibition or tumor metastasis inhibition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method of the present invention will be explained in detail in thefollowing.

In an anhydrous glutamic acid or derivatives thereof contained asessential component in a method of the present invention, anhydrousglutamic acid have the structure that glutamic acid isintramolecular-dehydrated and become circular as shown inabove-mentioned structural formula.

Similarly, in pyroglutamic acid (2-pyrrolidone-5-carbonxylic acid) orderivatives thereof contained as essential component in a method of thepresent invention, pyroglutamic acid also have the structure thatglutamic acid is intramolecular-dehydrated and become circular.

Both anhydrous glutamic acid and pyroglutamic acid concerning thepresent invention can be got by means of chemical synthesis, enzymaticmethod, or hydrolysis process, extraction process or the like fromnatural product. In all of these methods, L-glutamic acid, D-glutamicacid or DL-glutamic acid of optical isomer can be used as glutamic acid,but L-glutamic acid or DL-glutamic acid is preferable in view of effectof the invention, and, besides, L-glutamic acid is the most desirable.

The derivative anhydrous glutamic acid is hydrochloride salt ofanhydrous glutamic acid. The derivative of pyroglutamic acid is sodiumsalt of an anhydrous glutamic acid.

Anhydrous glutamic acid can be synthesized in accordance with well knownmethod (for example, J. Kollonitsch and A. Rosegay, Chemistry andIndustry, 7: 1867, (1964)). For instance, thionyl chloride is added intrifluoroacetic acid solution including L-glutamic acid, and dehydrationreaction is caused, and diethyl ether is added there to producesediment, and then under refrigeration anhydrous L-glutamic acid can beprepared by recrystallization using diethyl ether. In addition, variousderivatives can be synthesized chemically or enzymatically byconventional method using this anhydrous L-glutamic acid as startingmaterial.

Pyroglutamic acid and derivatives thereof can be synthesized chemicallyin accordance with well known method, for example, L-glutamic acid andwater (equal weight each other) is heat-treated at about 130-150 degreesCelsius in autoclave to prepare L-pyroglutamic acid, or heat-treated atabout 190-200 degrees Celsius to prepare DL-pyroglutamic acid byracemization. In addition, various derivative can be synthesizedchemically or enzymatically by conventional method using thisL-pyroglutamic acid or DL-pyroglutamic acid as starting material.

The following method can be used to prepare anhydrous glutamic acid ofthe present invention from natural product. Protein or peptide derivedfrom animals, plants, fish and shellfish are hydrolyzed by hydrochloricacid or protease etc. and further refined to prepare anhydrous glutamicacid. Fruit body of Basidiomycetes or mycelium is used preferably as rawmaterials. These are dried to give powder thing, or extract-processedusing extracting solvent to give extract solution or concentratedsolution, which are dried to give extract, and further separated orfractionated using organic solvent or adsorbent to prepare purifiedmaterial of high concentration.

In the present invention, these are used as preferred embodiment ofanhydrous glutamic acid and derivatives thereof.

When pyroglutamic acid is prepared from natural product, purifiedmaterial of high concentration can be prepare by the method same asanhydrous glutamic acid, these are used as preferred embodiment ofpyroglutamic acid and derivatives thereof.

In here, it is preferable to use one or more materials selected fromgroup comprising of Lentinus edodes, Flammulina velutipes, Lyophyllumaggregatum, Pleurotus ostreatus, Agaricus fungus, Phellinus linteusfungus, Ganoderma lucidum, Hericium Erinaceum fungus, Coriolusversicolor, Agaricus campestris, Grifola frondosa, Sparassis crispa,Schizophyllum commune, Tremella fuciformis Berkeley, and Cordycepssinensis (tochukaso) as Basidiomycetes. All of these fruit bodies ofmushroom are cultivated artificially or harvested abundantly, and are inthe marketplace, therefore there are available easily. These are eatenraw, or as dried material, the powder, or extract etc . . . . Inaddition, there are materials that polysaccharides included in extractlike Lentinus edodes, Coriolus versicolor, or Schizophyllum commune areused as drug. In the present invention, judging from desired effect, onekind or more kinds chosen among group comprising of Agaricus mushroom,Phellinus linteus, Ganoderma lucidum, Hericium Erinaceum fungus andCordyceps sinensis (tochukaso) are more preferable, and Agaricus fungusis the most desirable.

Agaricus mushroom is fungus of Agaricaceae and belongs to Agaricusgenus, and Agaricus blazei Murill or Agaricus bisporus can beillustrated. It is known that the former includes polysaccharide(beta-D-glucan) and polysaccharide protein conjugate and have antitumoraction and hypoglycemic action. Phellinus linteus is fungus ofHymenochaetaceae, and it is said that polysaccharide of the hotwater-extract shows anticancer action. Ganoderma lucidum is fungus ofPolyporaceae s. l. and also referred to as Ganoderma lucidum.Antiallergic action, antitumor action, and blood pressure stabilizationaction by terpenoid and polysaccharide, and hypoglycemic action etc. byproteoglycan are known. A Hericium erinaceum mushroom belongs toHericium ramosum (Merat) Banker, and then anticancer action byheterozygous beta-D-glucan component and active oxygen-erasing actionare known.

In the present invention, plain or dried fruit body of theBasidiomycetes can be used as raw materials. However, dried fruit bodyis preferable in view of handling, keeping quality and extractionefficiency. Plain or dried mycelium, which is prepared by incubation ofinoculm using culture medium including suitable carbon source andnitrogen source, can be used. And then, dried mycelium is convenientsame as case of the fruit body.

In addition, according to the present invention, mycelium can be used asraw materials, and culture broth generating in mycelium production canbe also used.

The culture broth is concentrated appropriately, and purified usingsolvents as described in the following.

In the present invention, it is characterized that anhydrous glutamicacid and derivatives thereof and pyroglutamic acid and derivativesthereof of the present invention are extracted by “water and/orhydrophilic organic solvent”, or “water and/or hydrophilic organicsolvent and hydrophobic organic solvent” from fruit body of theBasidiomycetes or mycelium. Desired effect of the present invention doesnot almost appear in extract that is extracted by only hydrophobicorganic solvent. Methanol, ethanol, n-propanol, isopropanol or acetoneis desirable for hydrophilic organic solvent, and hexane or chloroformis desirable for hydrophobic organic solvent. Hydrophilic organicsolvent can be used in mixture with water, and each of hydrophilicorganic solvent and hydrophobic organic solvent can be used in single ormix. The important thing on extraction of active ingredient ofcomposition of the present invention is following point. Morespecifically, extract including water-soluble component mainly is givenby using “water and/or hydrophilic organic solvent”, or mix solvent withhydrophobic organic solvent, and readily-soluble component such assaccharide or amino acid, which is more water-soluble, is separated andremoved by using hydrophilic organic solvent, and then oilinesscomponent such as lipid class is separated and removed by usinghydrophobic organic solvent.

When hydrophilic organic solvent and hydrophobic organic solvent aremixed, preferable mix ratio (volume ratio) is former/latter=9/1-1/9,more preferably, 5/1-1/5, and most preferably, 3/1-1/1. Extractionefficiency of essential component of the present invention deterioratespossibility and desired Effect is not provided possibility when mixratio gets out of the range. Extracting solvent is used in weight ofabout 3-20 times against dried material or extract of fruit body ormycelium. When it is less than 3 times, yield of extract is low. On thecontrary, if using a large quantity beyond 20 times, extractionefficiency does not improve more.

Extract solution is given by extraction process which is carried out,while fruit body of Basidiomycetes or mycelium come in contact with theextract solvent, for about 10 minutes to about 10 hours under normalpressure or pressurized, preferably 1 to 3 air pressure, at roomtemperature or around 100 degrees Celsius while stirring or reflowingappropriately. The solvent of the extract solution is removed byvacuum-dry, freeze-dry, or spray-dry etc. to prepare extract ofBasidiomycetes. In addition, the extract is fractionated by hydrophilicorganic solvent and hydrophobic organic solvent to give concentratewhich is further raised content of essential component of the presentinvention. Furthermore, the concentrate is run through columnchromatography with adsorbent such as silica gel, activated alumina,magnesium silicate, activated carbon, cellulose, or ion exchange resinto fractionate, and then purified material of high concentration can beprepared.

As above described, anhydrous glutamic acid, pyroglutamic acid, orderivatives thereof prepared by chemical synthesis or extract-methodfrom Basidiomycetes, and the extract solution, extract, concentrate, orpurified material including thereof, can be used for making compositionfor angiogenesis inhibition of the present invention with or withoutappropriate carrier, excipient, or additive. As far as, in compositionfor angiogenesis inhibition of the present invention, it is not againsta purpose of the present invention, various kinds of raw materials andcomponent are used together, for example, excipient, desiccant,antiseptic, nutrient, thickener, emulsifier, antioxidant, sweetener,acidulant, flavor enhancer, colorant, or flavor, which is used forconventional food and drug, as pharmaceutically acceptable carrier oredible carrier. In addition, it is one of the preferable aspects of thepresent invention to use well-known material having action inhibitingangiogenesis together.

In addition, composition of the present invention is also the thingwhich functions as composition for use in neoplasm inhibition orimmunostimulation, and prominent effect is shown. In other words,composition of the present invention acts as composition for use inprevention or treatment of a vascular-related disease, and shows,besides angiogenesis inhibitory action, antitumor action, namelyinhibition action of development and metastasis of tumors, and furtherimmunoenhancement action by oral ingestion etc . . . . In addition,there is effect for rheumatoid arthritis, diabetic retinopathy,psoriasis, neovascular glaucoma, inflammatory dermatosis, joint fluidrheumatism, osteoarthritis, atherosclerosis, obstructive affection suchas myocardial infarction. Therefore, the composition can be utilized ascomposition having above actions, and eating or drinking product, drug,pet food, or fodder for domestic livestock or domestic chickens etc. canbe illustrated for a concrete embodiment of the composition. Especially,eating and drinking product and drug are desirable.

As an embodiment of eating and drinking product, dry powder, extract, orpurified material of the Basidiomycetes, or the composition includingthem can be added in liquid, gel, powder, or solid food. For example,these can be added in fruit beverage, refreshing drinks, tea, soup,jelly, yogurt, pudding, cake mixture, furikake (powdered food to besprinkled over rice), miso (bean paste), soy sauce, dressing,mayonnaise, flavor enhancer such as dip of grilled beef, noodles,processed food of meat and fish such as ham or sausage, jam, cow milk,cream, powder, solid, or liquid milk-product such as butter or cheese,margarine, bread, cake, or cookie etc . . . .

In addition, these are processed into powder, granule, pellet, tabletalong with dextrin, lactose, starch or elaboration material thereof,excipient such as cellulose, vitamin, mineral, fat and oil of flora andfauna and fish and shellfish, protein, sugar, coloring agent, flavor,other above edible additive, if necessary, and these are covered ingelatine, and these are molded as capsule, and these are made to healthdrink usable as nutritional supplementary food and health food. Then,the composition that used well-known edible material having angiogenesisinhibition action together is preferred. In addition, eating anddrinking product of the present invention extends over extremely variouskinds of forms, it is not limited to these illustrations, but aboveforms of the nutritional supplementary food and health food aredesirable.

In the present invention, compounding dosage of composition of thepresent invention in eating and drinking product is hard to beprescribed uniformity because of differences in kind, form, or usepurpose of the eating and drinking product, and kind or form ofcomposition to combine, but when adding in general processed food, about0.01-50% by weight, more preferably 0.1-30% by weight on the basis ofanhydrous glutamic acid or pyroglutamic acid. When dosage gets out ofthe range and is under 0.01%, desired effect of the present invention byoral ingestion is small. On the contrary, if dosage is too much, aflavor is harmed potentially by kind of eating and drinking product, andthere is the case that it becomes impossible to prepare the eating anddrinking product.

In embodiments as drug of the present invention, above composition isadded well-known excipient or additive which is not against a purpose ofthe present invention, if necessary, and it was processed byconventional method to form preparation such as tablet, capsules,granulation, powder, or injection. By oral administration, enteraladministration, blood vessel administration, or intradermal injection,the composition can be used to develop at least one of effect ofangiogenesis inhibition, tumor inhibition, or immunostimulation, and thecomposition can be applied to prevent or treat various diseasesaccompanied by vascular neogenesis, propagation and transition of tumor,or reduction of immunocompetence. Combination dosage of composition ofthe present invention is hard to be prescribed uniformity because ofdifferences in kind, form, or use purpose of the preparation formedicine, but about 0.01-70% by weight, on the basis of anhydrousglutamic acid or pyroglutamic acid. In case of oral administration,dosage is not limited in particular, but it is 0.01-20 g, morepreferably 0.1-10 g as base in anhydrous glutamic acid or pyroglutamicacid per adult (50 kg in weight)/1 day. When dosage gets out of therange and is under 0.01 g, desired effect of the present invention issmall. On the contrary, if dose is too much, prominent effect cannot beexpected more.

Example 1

Dry fruit body of an agaricus mushroom (Agaricus blazei Murill) wascrushed. It was then added with chloroform/methanol=1/1 mixed solutionafter which the mixture was warmed to 40 degrees Celsius, andextract-processed for one hour to give a chloroform/methanol=1/1extract. Methanol was added to the extract, and methanol soluble layerwas separated and collected. Furthermore, hexane was added in themethanol soluble layer, and hexane insoluble layer (sample 1) wascollected. The hexane insoluble layer was then run through a silica gelcolumn chromatography (silanised Silicagel 60 PF 256: Merck 7751,water/methanol=7/3), and ninhydrin reaction positive fractions (fractionNo. 5 and 6) were collected. The fractions (fraction No. 5 and 6) werethen run through HPLC (ShimpakPREP-ODS (M), column: 20φ×250 mm,Shimadzu, RT, 6 ml/min, water/methanol=5/1), and ninhydrin reactionpositive fractions (Rt=6 to 12 min) were collected. The fractions (Rt=6to 12 min) were further run through HPLC (ShimpakPREP-ODS (M), column:20φ×250 mm, Shimadzu, RT, 6 ml/min, water), and ninhydrin reactionpositive fraction (Rt=8 to 22 min) were collected. These ninhydrinreaction positive fractions were further refined by running through TLC(Silicagel 60 PF 254:Merck7747, water/methanol=1/100), and it wasensured that an anhydrous glutamic acid was included (Rf=0.41). Inaddition, results of mass spectrum and nuclear magnetic resonance (NMR)analysis showed that the anhydrous glutamic acid was L-optical isomer.

Example 2

By means of culture apparatus “JARFERMENTER” of 10 liters capacity,while aerating (2 vvm), inoculum culture broth of Phellinus linteus (1liter) was incubated in culture medium including glucose (5% by weight),yeast extract (0.5% by weight) and peptone (2.0% by weight) at 28degrees Celsius for 72 hours, and culture mycelium (165 g) wascollected. The mycelium was dried and crushed to give mycelium powder. Ahexane/ethanol/water=2/3/1 mixed solvent was then added to the myceliumpowder after which the mixture was extract-processed at room temperaturefor three hours to give an extract (sample 2). The extract was furtherfractionation-processed by ethanol, and an ethanol soluble layer wasseparated and collected. The ethanol soluble layer was thenfractionation-processed by hexane, and hexane insoluble layer wascollected. The hexane insoluble layer was fractionated and purified bysilica gel column chromatography, HPLC, and TLC as same as example 1.Existence of anhydrous L-glutamic acid was ensured from results of massspectrometry and nuclear magnetic resonance assay.

Example 3

In accordance with a method as described in the above documents, ananhydrous L-glutamate was synthesized. More specifically,trifluoroacetic acid 75 ml and L-glutamic acid mol were added tofour-mouth flask, and the mixture was stirred to dissolve. Whilestirring the solution, thionyl chloride 0.28 mol was dripped slowly intothe flask. 30 minutes later of end of dripping, diethyl ether 35 ml wereadded slowly into the flask to produce sediment. After holding to 5degrees Celsius for one hour, diethyl ether 100 ml were added to repeatrecrystallization. An anhydrous L-glutamate acid hydrochloride (sample3) was then prepared.

Example 4

An anhydrous DL-glutamate acid hydrochloride (sample 4) was preparedusing the same way as in example 3 except that DL-glutamic acidsubstituted for L-glutamic acid of raw materials.

Example 5

Dry fruit body of Agaricus blazei Murill was crushed, and water wasadded there to extract-process using hot water at 80-95 degrees Celsiusby conventional method. The extract was then dried under reducedpressure to prepare a hot water-extract of agaricus fungus. Ethanolmultiplied by three (weight) was added to 40% by weight aqueous solutionof the hot water-extract, and after mixing, ethanol layer was collected.The ethanol layer was then dried under reduced pressure to prepareethanol soluble material (sample 5).

Comparative Example 1

Dry fruit body of Agaricus blazei Murill, and water was added toextract-process using hot water at 80-95 degrees Celsius by conventionalmethod. The extract was then dried under reduced pressure to prepare ahot water-extract of agaricus fungus corresponding to marketing product(reference sample 1).

Test Example 1

Angiogenesis inhibition action of anhydrous glutamic acid, itsderivatives, and various processed materials containing them concerningthe present invention was examined by degree of angiogenesis induced bymeans of MATRIGEL™ matrix (made by Becton Dikinson Labware Company, itis “cell culture backing material”, following “MATRIGEL”.) according toa method of Passaniti et al (Laboratory Invest., Vol. 67, Page 519-528,1992).

An example of the MATRIGEL matrix is shown with the following: BDMatrigel Basement Membrane Matrix is a solubilized basement membranepreparation extracted from the Engelbreth-Holm-Swarm (EHS) mousesarcoma, a tumor rich in ECM proteins. Its major component is laminin,followed by collagen IV, heparan sulfate proteoglycans, entactin andnidogen.35 It also contains TGF-β fibroblast growth factor, tissueplasminogen activator36 and other growth factors which occur naturallyin the EHS tumor. At room temperature, BD Matrigel Matrix polymerizes toproduce biologically active matrix material resembling the mammaliancellular basement membrane.

More specifically, normal mice (5 mice/1 group) were used after breedingC57BL/6 female mice (5 week old, purchased from Charles River JAPAN,Inc.) preliminarily for one week. While cooling test materials as shownin the following, each of the test materials 0.5 ml were transplanted tosubcutaneous of abdomen of the mice. MATRIGEL was taken out on the sixthday after transplantation, and state of angiogenesis was observed. Inaddition, the MATRIGEL was freeze-dried, and it was estimated theweight. Furthermore, pure water 1 ml was added to the MATRIGEL. Afterhomogenizing by Polytron and centrifuging at 2000 rpm for five minutes,supernatant was filtrated through filter with 0.2 μm and hemoglobindosage was measured by using Hemoglobin-TestWako™ (made by Wako PureChemical Industries, Ltd.).

Group 0 of nothing addition: MATRIGEL

Control group: MATRIGEL, heparin (64 units), Acidic Fibroblast GrowthFactor (it is abbreviated to a-FGF as follows.) (1 ng/ml)

Group 1 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 3 (800 μg/ml)

Group 2 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 3 (400 μg/ml)

Group 3 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 3 (200 μg/ml)

Group 4 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 4 (800 μg/ml)

Group 5 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 1 (600 μg/ml)

Group 6 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 2 (800 μg/ml)

Group 7 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample1+sample 3 (for each 200 μg/ml)

Group 8 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 5 (600 μg/ml)

Group 9 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), reference sample 1 (800 μg/ml)

A result of the test was shown in Table 1 and Table 2. The numericalvalues of each table were displayed in n=5, average value±standarderror. As is apparent from each table, in control group, angiogenesiswas promoted remarkably and weight of MATRIGEL and hemoglobin dosagewere increased compared with a group 0 of nothing addition. On the otherhand, in groups of test material addition, augmentation of weight ofMATRIGEL and hemoglobin dosage were concentration-dependent controlledat the group using sample 3 (anhydrous L-glutamate acid hydrochloride),and these results showed excellent angiogenesis inhibition effect. Thegroup using sample 4 (anhydrous DL-glutamate acid hydrochloride) wasalso recognized an effect, which is slightly low, but almost equal tosample 3, of angiogenesis inhibition. In addition, sample 1(purification material of agaricus mushroom extract) and sample 2(extract of Phellinus linteus) also showed strong angiogenesisinhibition effect. However, the angiogenesis inhibition effect was smallwith reference sample 1 (hot water extract of an agaricus mushroom).

TABLE 1 MATRIGEL Matrix No. Group material (μg/mL) weight (mg) n = 5 1Group 0  77.7 ± 10.1 (Normal) 2 Control 413.3 ± 42.5 3 Group 1 sample 3(800) 110.9 ± 11.4 4 Group 2 sample 3 (400) 232.2 ± 52.7 5 Group 3sample 3 (200) 276.6 ± 27.6 6 Group 4 sample 4 (800) 237.3 ± 30.1 7Group 5 sample 1 (600) 105.0 ± 12.3 8 Group 6 sample 2 (800) 149.4 ±24.7 9 Group 7 sample 1/sample 3 = 1/1 167.3 ± 33.2 (400) 10 Group 8sample 5 (600) 181.1 ± 20.2 11 Group 9 reference sample 1 (800) 357.5 ±36.2

TABLE 2 Quantity of No. Group material (μg/mL) hemoglobin (mg) n = 5 1Group 0  4.6 ± 1.0 (Normal) 2 Control 30.4 ± 2.6 3 Group 1 sample 3(800)  3.9 ± 0.5 4 Group 2 sample 3 (400) 15.5 ± 4.6 5 Group 3 sample 3(200) 21.5 ± 4.0 6 Group 4 sample 4 (800) 13.0 ± 2.9 7 Group 5 sample 1(600)  4.0 ± 0.8 8 Group 6 sample 2 (800)  5.4 ± 0.7 9 Group 7 sample1/sample 3 = 1/1  9.3 ± 2.2 (400) 10 Group 8 sample 5 (600)  7.6 ± 2.411 Group 9 reference sample 1 (800) 27.3 ± 3.7

Test Example 2

Antiproliferative action of tumor and metastasis inhibition action oftumor were examined and evaluated by the following method about samplesconcerning the present invention. The Lewis lung cancer (it isabbreviated to LLC as follows) cell which was sold in lots fromInstitute of Physical and Chemical Research, was suspended to phosphoricacid/physiology salt buffer solution (pH 7.4). On the other hand, normalmice (7 mice/1 group) were used after breeding C57BL/6J female mice (6week old, purchased from Kurea Japan, Inc.) preliminarily for one week.Under nembutal anesthesia the mice were exposed spleen through a smallincision, and after injecting the LLC cell suspension (number of the LLCcell: 1.0×10⁵) into the exposed spleen, the small incision was sewed uppromptly. After 12 hours of LLC cell transplantation, the mice wereorally administered agaricus mushroom extract (sample 1) 100 mg/kg (bodyweight) or 300 mg/kg (body weight) once a day for 20 days in succession.Distilled water was administered to a normal group and control group(LLC tumor-bearing mice) instead of sample 1. During this test period,the propagation degree of cancer cell was measured every two or threedays by measuring quantity of carcinoma tissue volume (it was calculatedwith (major axis)×(minor axis)²/2). On the 21st day after cancer celltransplantation, under ether anesthesia, mice of each group were takenvenous blood sample into heparin tube, and white blood cell count, redblood cell count and hemoglobin dosage in the blood were measured by acall counter of blood cell. In addition, mice were slaughtered aftercollection of blood, and carcinoma tissue, liver, lung, spleen andthymus gland were removed and weighed, and then the number of cancercell colony which spread to pulmonary tissue was measured understereoscopic microscope.

Quantity of volume of tumor tissue in the LLC cell transplantation miceare shown in Table 3, weight of carcinoma tissue and each organ areshown in Table 4, and white blood cell count, red blood cell count,hemoglobin dosage and the number of transition colony to lung are shownin Table 5. Numerical values in each table are shown by averagevalue±standard error. Fisher's Protect LSD Test was executed forsignificant difference assay, using a significant difference (P<0.05).

TABLE 3 Group (n = 7) test group1 test group2 Material (mg/kg) controlsample 1 (100) sample 1 (300) quantity of neoplasm volume (mm³) aftertransplantation  5 days 362 ± 33  290 ± 40   164 ± 30 (*)  8 days 461 ±94  307 ± 64   247 ± 52 (*) 10 days 538 ± 108 322 ± 57 (*)  301 ± 73 (*)14 days 745 ± 142 493 ± 115    323 ± 101 (*) 17 days 872 ± 173 517 ± 121(*)  346 ± 108 (*) 20 days 1505 ± 362  608 ± 133 (*) 410 ± 89 (*) (*):There was a significant difference in comparison with control group (P <0.05)

TABLE 4 Group (n = 7) test group1 test group2 Material (mg/kg) normalcontrol sample 1 (100) sample 1 (300) First body weight (g) 17.9 ± 0.3018.2 ± 0.21 18.0 ± 0.23 17.8 ± 0.25 Last body weight (g) 21.0 ± 0.4119.9 ± 0.54 20.3 ± 0.72 20.0 ± 0.24 Neoplasm weight (mg) — 1872 ± 604  531 ± 267*  460 ± 115* Spleen (g)  0.07 ± 0.01* 0.71 ± 0.03  0.35 ±0.02*  0.22 ± 0.02* Liver (g) 1.18 ± 0.05 1.28 ± 0.04 1.22 ± 0.03 1.24 ±0.06 Lung (mg) 152.8 ± 4.51  166.8 ± 9.20  165.0 ± 6.76  155.0 ± 5.14 Thymus gland (mg) 47.0 ± 5.3  41.6 ± 10.3 45.2 ± 4.2  50.5 ± 3.0  *Therewas a significant difference in comparison with control group (P < 0.05)

TABLE 5 Group (n = 7) test group1 test group2 Material (mg/kg) normalcontrol sample 1 (100) sample 1 (300) White blood cell count 3.65 ± 0.344.85 ± 0.30 4.08 ± 0.54 4.30 ± 0.79 (×10³/μL) (*) (*) (*) Red blood cellcount 782.0 ± 21.0  494.2 ± 30.4  631.5 ± 26.2  699.2 ± 51.5  (×10⁴/μL)(*) (*) (*) Quantity of hemoglobin 11.70 ± 1.09  6.93 ± 0.72 9.62 ± 0.4310.51 ± 0.54  (g/100 mL) (*) (*) Number of transition — 21.5 ± 2.5  10.0± 1.5  7.0 ± 1.0 colony in lung (*): There was a significant differencein comparison with control group (P < 0.05)

From data of table 3, it was showed that volume of neoplasm increasedwith time in control group (tumor-bearing mice) by transplantation ofthe LLC cell, and that increase of volume of neoplasm was inhibited inthe group administered orally the test material (sample 1: agaricusmushroom extract including anhydrous glutamic acid) and the LLC cellularpropagation was controlled.

From data of table 4, it was showed that increase of weight of neoplasmwas inhibited clearly by intake of the test material, and that there wasno significant difference in weight of each organ aside from spleen andfinal body weight between normal group, LLC cell transplantation group(control group) and test material administrated group.

Spleen weight increased in control group, but depression of increase wasrecognized in test material administrated group (there was significancedifference with P<0.05). Therefore, it was proved that propagation ofLLC cell was depressed by oral ingestion of test material (sample 1).

From data of table 5, it was showed that white blood cell countincreased in control group compared with normal group, and that therewas no significant difference between test material administrated groupand normal group. Moreover, it was showed that red blood cell count andquantity of hemoglobin were significantly reduced and anemia state wasshown in control group, but in test material administrated group, theywere increased in significance (P<0.05) and anemia state recovered toclose to normal by means of oral administration of agaricus fungusextract (sample 1). In addition, the number of transition colony of LLCcell in lung decreased in significance in test material administratedgroup compared with control group, and then it was proved thattransition of cancer cell was inhibited by oral ingestion of theagaricus fungus extract which contained anhydrous glutamic acid (sample1).

Test Example 3

Effect on immune function was examined and evaluated by the followingmethod about samples concerning the present invention. Splenocyte wasseparated from spleen removed with test example 2. The splenocyte waslayered on lymphocyte separate solution (“Lymphocyte SegregationSolution”, made by Dainippon Pharmaceutical Co., Ltd.) and centrifugedfor 30 minutes at 2000 rpm to separate lymphocyte. The coexistingerythrocyte was then hypotonic-solutionized and removed. The number ofthe lymphocyte was measured and adjusted to 1×10⁶ cell count/100 μL.Antibody of various cell surface antigen (“CD 4, antimouse, FITC label”,“CD 8, antimouse, FITC label” and “NK1.1, antimouse, R-PE label”, madeby Dainippon Pharmaceutical Co., Ltd.) 10 μL were added to thelymphocyte and reacted at 4 degrees Celsius for 30 minutes, after whichit was washed twice in phosphate buffer (reagent for biochemicalanalysis made by Wako Pure Chemical Industries, LTD). The phosphatebuffer was added there so that total volume was 1 mL, and then thenumbers of CD 4⁺, CD 8⁺ and NK1.1⁺ T cell were measured using a flowcytometry. The results are shown in Table 6.

TABLE 6 Group (n = 7) test group1 test group2 Material (mg/kg) normalcontrol sample 1 (100) sample 1 (300) Number of the lymphocyte (*) (*)(*) (×10⁷/spleen) 3.68 ± 0.34 1.23 ± 0.20 3.15 ± 0.41 2.98 ± 0.90 Numberof CD4⁺T CELL (*) (*) (×10⁶/spleen) 6.0 ± 1.5 2.0 ± 0.5 3.5 ± 1.0 5.5 ±1.0 Number of CD8⁺T CELL (*) (*) (*) (×10⁶/spleen) 8.1 ± 1.0 3.5 ± 0.66.7 ± 0.7 6.5 ± 1.0 Number of NK1.1⁺T CELL (*) (*) (×10⁵/spleen) 2.0 ±0.5 2.0 ± 0.3 4.5 ± 0.5 3.5 ± 0.5 (*): There was a significantdifference in comparison with control group (P < 0.05)

From data of table 6, it was showed that the number of the lymphocyte inspleen decreased in significance in control group (tumor-bearing mice)compared with normal group, and that this decrease was controlled intest material administrated group. In addition, it was proved that thenumbers of CD 4⁺ T cell and CD 8⁺ T cell in spleen decreased insignificance in control group (tumor-bearing mice) compared with normalgroup, and that decrease of the numbers of both cell was inhibited andthe number of NK1.1⁺ T cell was increased by intake of test material(sample 1). From these finding, it became clear that immune function wasreinforced by oral ingestion of agaricus mushroom extract includinganhydrous glutamic acid.

Example 6

Angiogenesis inhibition composition of the present invention comprisingsample 1 and oolong tea leaf powder (3:2 (a weight ratio)) 5.0 kg,modified starch (a product made by Matsutani Chemical Industry Co, .Ltd,brand name: pine flow) 3.5 kg, tricalcium phosphate 0.3 kg, vitamin B₁0.3 kg, vitamin B₂ 0.2 kg, vitamin B₆ 0.2 kg, and vitamin C 0.5 kg weretucked into blender and mixed for 10 minutes. After the mixture wassupplied into a tablet machine of the direct compression formulationtype and made a tablet (diameter of 7 mm, height of 4 mm, 150 mg inweight), food in tablet form was produced experimentally by coating withshellac thin-film using coating machine. This tablet can be used forpurpose of enhancement of internal immune strength, and prevention oflifestyle-related diseases such as diabetes mellitus or carcinoma.

Example 7

Butter 110 g, shortening 110 g, very-refined sugar 90 g and milk 100 mLwere put in bowl for families, and one egg was more added there whilewhipping together. After mixing enough, a mixture of sample 2 and sample3 (3:1, weight ratio) of the present invention 10 g was added therealong with soft flour 190 g and baking powder 2 g, and then the mixturewas kneaded enough together. After setting dough for 30 minutes, themixed compound was split into 50 using mould and burnt in oven, and thena butter cookie was produced experimentally.

Example 8

Composition of the present invention comprising of sample 1, sample 2,and grape seed extract (made by Interhealthl company, brand name:Activin) (1:2:1 (a weight ratio)) 5 g were added to a commercialvegetable juice 1 L and mixed together, and then angiogenesis inhibitionvegetable juice was produced experimentally for a person worried aboutmalignant growth or rheumatoid arthritis. There was no inferiority inthis in comparison with original vegetable juice at all.

Example 9

A mixture of sample 3/sample 5=1/1 (a weight ratio) 130 kg, propolis 90kg, yellow beeswax 15 kg and corn oil 150 kg were mixed enough to becomehomogeneous liquid thing while warming to 40 degrees Celsius. This wassupplied to the capsule filling up machine, and then gelatinecovered-capsule formulation, of which quantity of one grain was 250 mg,was produced experimentally. This formulation can be used as ediblecomposition (food and drink) or composition for medicine (drug) thatoral ingestion is possible.

Example 10

Dry fruit body of an agaricus mushroom (Agaricus blazei Murill) wascrushed. It was then added with chloroform/methanol=1/1 mixed solutionafter which the mixture was warmed to 50 degrees Celsius, andextract-processed for one hour to give a chloroform/methanol=1/1extract. Methanol was added to the extract, and then methanol solublelayer was separated and collected except methanol insoluble materialsincluding mannitol. Furthermore, hexane was added in the methanolsoluble layer, and then hexane insoluble layer (sample 6) was collected.The hexane insoluble layer was then run through a silica gel columnchromatography (silanised Silicagel 60 PF 254:Merck 7751,water/methanol=7/3), and ninhydrin reaction positive fractions (fractionNo. 5 and 6) were collected. The fractions (fraction No. 5 and 6) werethen run through HPLC (Shimadzu LC-8A system: Shimpak PREP-ODS (M),column: 20φ×250 mm, Shimadzu, RT, 6 ml/min, water/methanol=5/1), andninhydrin reaction positive fractions (Rt=6 to 12 min) were collected.The fractions (Rt=6 to 12 min) were further run through HPLC (ShimadzuLC-8A system: Shimpak PREP-ODS (M), column: 20φ×250 mm, Shimadzu, RT, 6ml/min, water), and ninhydrin reaction positive fraction (Rt=8 to 22min) were collected. These ninhydrin reaction positive fractions werefurther refined by running through TLC (Silicagel 60 PF 254:Merck7747,water/methanol=1/100), and it was ensured that materials shown in thefollowing were included.

More specifically, as a result of TLC (Silicagel 60, precoat TLC:Merck5715, water/methanol=1/100) assay, it was showed that there werealanine (the neighborhood of Rf (rate of flow)=0.27, ninhydrinpositivity), proline (the neighborhood of Rf=0.20, ninhydrinpositivity), gamma aminobutyric acid (the neighborhood of Rf=0.15,ninhydrin positivity) and unknown substance (the neighborhood ofRf=0.40, ninhydrin negativity) (49:5:25:21 (a weight ratio)).Subsequently, the unknown material was analyzed by NMR (nuclear magneticresonance) spectrum analysis (device: Varian Unity Inova 500) and massspectrum analysis (device: M-4000H, made by Hitachi, Ltd.), the resultswere 1H-NMR spectra (δ ppm, D₂O): 2.09, 2.39 (each 1H, m, H-3), 2.50(2H, m, H-4), and 4.22 (1H, dd, J=5.2 and 9.0 Hz), 13C-NMR spectra (δppm, D₂O): 184 (COOH or —C═O—), 182 (—C═O—), 57 (—CH—), 32 (—O═C—CH₂—),and 28 (—CH₂—CH₂—), and mass spectrum (m/z): 42, 84 and 129 (M+). Inaddition, the result of Optical Rotatory Dispersion (ORD) spectrumanalysis (made by JASCO Corporation (NIHON BUNKOU), ORD/UV-820) was[alpha]_(D) ²³ −11.5° (c=2, H₂O). From these assay results, the unknownmaterial was identified as L-pyroglutamic acid.

Example 11

By means of culture apparatus of 10 liters capacity, while aerating (2vvm) and stirring (150 rpm), inoculum culture broth of Phellinus linteus(1 liter) was incubated in culture medium including glucose (5% byweight), yeast extract (0.5% by weight) and poly peptone (1.5% byweight) at 26 degrees Celsius for seven days, and culture mycelium (200g) was collected. The culture mycelium was dried and crushed to givemycelium powder. A hexane/ethanol/water=3/4/1 mixed solvent was thenadded to the mycelium powder after which the mixture was warmed to 40degrees Celsius, and extract-processed for 30 minutes to give an solublematerial (sample 7). The soluble material was furtherfractionation-processed by ethanol, and an ethanol soluble layer wasseparated and collected. The ethanol soluble layer was thenfractionation-processed by hexane, and hexane insoluble layer wascollected. The hexane insoluble layer was fractionated and purified bysilica gel column chromatography, HPLC, and TLC as same as example 10.Existence of L-pyroglutamic acid was ensured from results of massspectrometry and nuclear magnetic resonance assay etc.

Example 12

L-pyroglutamic acid sodium was prepared in accordance with conventionalmethod. More specifically, L-pyroglutamic acid aqueous solution (30% byweight) was added to a flask with stirrer, and while stirring slowly atroom temperature, sodium hydroxide aqueous solution (0.5N) was addedthere untill no pH fluctuation. After salting out, the solution wasdried to give L-pyroglutamic acid sodium (sample 8).

Example 13

DL-pyroglutamic acid sodium (sample 9) was prepared using the same wayas in example 12 except that DL-glutamic acid substituted for L-glutamicacid of raw materials.

Example 14

Dry fruit body of Agaricus blazei Murill was crushed, and water wasadded there to extract-process using hot water at 80-95 degrees Celsiusby conventional method. The extract was then dried under reducedpressure to prepare a hot water-extract of agaricus fungus. Ethanolmultiplied by three (weight) was added to 40% by weight aqueous solutionof the hot water-extract, and after mixing, ethanol layer was collected.The ethanol layer was then dried under reduced pressure to give ethanolsoluble material. The ethanol soluble material was washed by hexane of 5times (weight), and then dried under reduced pressure to prepare hexaneinsoluble material (sample 10).

Test Example 4

Angiogenesis inhibition action of pyroglutamic acid, its derivatives,and various processed materials containing them concerning the presentinvention was examined by degree of angiogenesis induced by means ofMATRIGEL™ matrix (Becton Dikinson Labware company, following “MATRIGEL”)according to the same method as test example 1.

More specifically, normal mice (5 mice/1 group) were used after breedingC57BL/6 female mice (5 week old, purchased from Charles River JAPAN,Inc.) preliminarily for one week. While cooling test materials as shownin the following, each of the test materials 0.5 ml were transplanted tosubcutaneous of abdomen of the mice. MATRIGEL was taken out on the sixthday after transplantation, and state of angiogenesis was observed. Inaddition, the MATRIGEL was freeze-dried, and it was weighed.Furthermore, pure water 1 ml was added to the MATRIGEL. Afterhomogenizing by Polytron and centrifuging at 2000 rpm for five minutes,supernatant was filtrated through filter with 0.2 μm and hemoglobindosage was measured by using Hemoglobin-TestWako™ (product made in WakoPure Chemical Industries, Ltd.).

Group 0 of nothing addition: MATRIGEL

Control group: MATRIGEL, heparin (64 units), Acidic Fibroblast GrowthFactor (it is abbreviated to a-FGF as follows.) (1 ng/ml)

Group 1 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 8 (800 μg/ml)

Group 2 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 8 (400 μg/ml)

Group 3 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 8 (200 μg/ml)

Group 4 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 9 (800 μg/ml)

Group 5 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 6 (600 μg/ml)

Group 6 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 7 (800 μg/ml)

Group 7 of test material addition: MATRIGEL, heparin (64 unit), a-FGF (1ng/ml), sample 6+sample 8 (for each 200 μg/ml)

Group 8 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), sample 10 (600 μg/ml)

Group 9 of test material addition: MATRIGEL, heparin (64 units), a-FGF(1 ng/ml), reference sample 1 (800 μg/ml)

A test result was shown in Table 7.

The numerical values of Table 7 were displayed in n=5, averagevalue±standard error.

As is apparent from Table 7, in control group, angiogenesis was promotedremarkably and weight of MATRIGEL and hemoglobin dosage were increasedcompared with a group 0 of nothing addition.

On the other hand, in groups of test material addition, augmentation ofweight of MATRIGEL and hemoglobin dosage was concentration-dependentcontrolled at the group using sample 8 (L-pyroglutamic acid salt), andthese results showed excellent angiogenesis inhibition effect. The groupusing sample 9 (DL-pyroglutamic acid salt) was also recognized aneffect, which is slightly low, but almost equal to sample 8, ofangiogenesis inhibition. In addition, sample 6 (purification material ofagaricus mushroom extract) and sample 7 (extract of Phellinus linteus)also showed strong angiogenesis inhibition effect. However, theangiogenesis inhibition effect was small with reference sample 1 (hotwater extract of an agaricus mushroom). Furthermore, presence ofangiogenesis inhibitory effect was also examined about alanine, prolineand gamma aminobutyric acid similarly, but the angiogenesis inhibitoryeffect was not recognized in these materials.

TABLE 7 Quantity Group MATRIGEL of hemoglobin n = 5 material (μg/mL)weight (mg) (mg/MATRIGEL) Group 0 100.0 ± 12.1  5.3 ± 1.7 (Normal)Control 393.6 ± 22.7 33.0 ± 3.4 Group 1 sample 8 (800) 121.5 ± 28.1  4.8± 1.2 Group 2 sample 8 (400) 190.2 ± 30.4 17.5 ± 3.6 Group 3 sample 8(200) 265.3 ± 18.6 23.4 ± 2.0 Group 4 sample 9 (800) 209.0 ± 30.8 15.8 ±2.7 Group 5 sample 6 (600) 110.7 ± 10.3  4.0 ± 0.5 Group 6 sample 7(800) 136.4 ± 24.5  6.1 ± 2.4 Group 7 sample 6/sample 8 = 1/1 150.5 ±23.1  8.3 ± 1.8 (400) Group 8 sample 10 (600) 154.1 ± 27.2  9.0 ± 4.3Group 9 reference 320.4 ± 35.2 30.5 ± 4.5 sample 1 (800) MATRIGEL ™(Becton Dikinson Labware company) matrix

Test Example 5

Antiproliferative action of tumor and metastasis control action of tumorwere examined and evaluated according to the same method as Test example2 except the following condition. 7 mice/1 group was replaced with 8mice/1 group. Sample 1 was replaced with sample 6. The period ofadministration was 30 days instead of 20 days. The propagation degree ofcancer cell was measured every 3 to 5 days instead of every 2 to 3 days.Slaughter of mice was conducted on the 31st day after cancer celltransplantation, instead of the 21st day.

Quantity of volume of tumor tissue in the LLC cell transplantation miceare shown in Table 8, weight of carcinoma tissue and each organ areshown in Table 9, and white blood cell count, red blood cell count,hemoglobin dosage and the number of transition colony to lung are shownin Table 10. Numerical values in each table are shown by averagevalue±standard error. Fisher's Protect LSD Test was executed forsignificant difference assay, using a significant difference (P<0.05).

TABLE 8 Group (n = 8) test group1 test group2 Material (mg/kg) controlsample 6 (100) sample 6 (300) quantity of neoplasm volume (mm³) aftertransplantation  7 days 405 ± 40 285 ± 72   182 ± 31 (*)  12 days 617 ±84 310 ± 94 (*)  244 ± 83 (*)  17 days  806 ± 137 552 ± 108   317 ± 105(*) 21 days 1430 ± 280 503 ± 131 (*) 416 ± 122 (*) 24 days 1916 ± 325745 ± 186 (*) 503 ± 169 (*) 30 days 2190 ± 382 1067 ± 292 (*)  670 ± 213(*) (*): There was a significant difference in comparison with controlgroup (P < 0.05)

TABLE 9 Group (n = 8) test group1 test group2 Material (mg/kg) normalcontrol sample 6 (100) sample 6 (300) First body weight (g) 18.5 ± 0.2518.3 ± 0.34 18.5 ± 0.26 18.2 ± 0.22 Last body weight (g) 21.4 ± 0.3623.1 ± 0.54 21.6 ± 0.87 22.1 ± 0.43 Neoplasm weight (mg) — 3061 ± 785 745 ± 403 480 ± 130 Spleen (g)  0.08 ± 0.02* 1.43 ± 0.07  0.81 ± 0.10* 0.45 ± 0.03* Liver (g) 1.21 ± 0.05 1.39 ± 0.10 1.25 ± 0.05 1.22 ± 0.04Lung (mg) 164.8 ± 7.1  187.7 ± 15.2  184.4 ± 13.1  175.0 ± 5.2  Thymusgland (mg) 58.5 ± 3.9  46.8 ± 6.6  47.9 ± 4.9  51.5 ± 5.0  (*): Therewas a significant difference in comparison with control group (P < 0.05)

TABLE 10 Group (n = 8) test group1 test group2 Material (mg/kg) normalcontrol sample 6 (100) sample 6 (300) White blood cell count (*1)(×10³/μL) 3.32 ± 0.18 5.73 ± 1.40 4.72 ± 1.13 4.12 ± 0.59 Red blood cellcount (*) (*) (*) (×10⁴/μL) 799.0 ± 7.4  506.3 ± 78.4  692.6 ± 37.2 743.2 ± 22.0  Quantity of hemoglobin (*) (*) (*) (g/100 mL) 12.4 ± 0.097.80 ± 1.25 10.6 ± 0.59 11.3 ± 0.39 Number of transition (*) (*) colonyin lung — 30.0 ± 2.8  15.8 ± 2.5  13.6 ± 1.4  (*): There was asignificant difference in comparison with control group (P < 0.05) (*1):There was a significant difference in comparison with normal group (P <0.05)

From data of table 8, it was showed that volume of neoplasm increasedwith time in control group (tumor-bearing mice) because oftransplantation of the LLC cell, and that increase of volume of neoplasmwas inhibited in the group administered orally the test material (sample6: agaricus mushroom extract including pyroglutamic acid) and the LLCcellular propagation was controlled.

From data of table 9, it was showed that increase of weight of neoplasmwas inhibited clearly by intake of the test material, and that there wasno significant difference in weight of each organ aside from spleen andfinal body weight between normal group, LLC cell transplantation group(control group) and test material administrated group. Spleen weightincreased in control group, but depression of increase was recognized intest material administrated group (there was significance differencewith P<0.05). Therefore, it was proved that propagation of LLC cell wasdepressed by oral ingestion of test material (sample 6).

From data of table 10, it was showed that white blood cell countincreased in significance in control group compared with normal group,and that there was no significant difference between test materialadministrated group and normal group. Moreover, it was showed that redblood cell count and quantity of hemoglobin were significantly reducedand anemia state was shown in control group, but in test materialadministrated group, they were increased in significance (P<0.05) andanemia state recovered to close to normal by means of oraladministration of agaricus fungus extract (sample 6). In addition, thenumber of transition colony of LLC cell in lung decreased insignificance in test material administrated group compared with controlgroup, and then it was proved that transition of cancer cell wasinhibited by oral ingestion of the agaricus fungus extract whichcontained pyroglutamic acid (sample 6).

Test Example 6

Effect on immune function was examined and evaluated by the followingmethod about samples concerning the present invention. Splenocyte wasseparated from spleen removed with test example 5. The splenocyte wasprocessed according to the same method as test example 3, and then thenumbers of CD 4⁺, CD 8⁺ and NK1.1⁺ T cell were measured using a flowcytometry. The results are shown in Table 11.

TABLE 11 Group (n = 8) test group1 test group2 Material (mg/kg) normalcontrol sample 6 (100) sample 6 (300) Number of the lymphocyte (*) (*)(*) (×10⁷/spleen) 3.52 ± 0.48 1.47 ± 0.22 2.84 ± 0.36 3.01 ± 0.45 Numberof CD4⁺T CELL (*) (*) (×10⁶/spleen) 6.5 ± 0.8 2.1 ± 0.4 4.6 ± 0.5 6.0 ±1.1 Number of CD8⁺T CELL (*) (*) (*) (×10⁶/spleen) 7.7 ± 1.2 4.5 ± 0.76.6 ± 0.6 8.9 ± 0.8 Number of NK1.1⁺T CELL (*) (*) (*) (×10⁵/spleen) 1.7± 0.2 2.6 ± 0.3 2.8 ± 0.3 4.0 ± 0.5 (*): There was a significantdifference in comparison with control group (P < 0.05)

From data of table 11, it was showed that the number of the lymphocytein spleen decreased in significance in control group (tumor-bearingmice) compared with normal group, and that this decrease was controlledin test material administrated group. In addition, it was also provedthat the numbers of CD 4⁺ T cell and CD 8⁺ T cell in spleen decreased insignificance in control group (tumor-bearing mice) compared with normalgroup, and that decrease of the numbers of both cell was inhibited andthe number of NK1.1⁺ T cell was increased by intake of test material(sample 6). From these finding, it became clear that immune function wasreinforced by oral ingestion of agaricus mushroom extract includinganhydrous glutamic acid.

Example 15

Angiogenesis inhibition composition of the present invention comprisingsample 10, oolong tea leaf powder, and Guava leaf hot water extract(3:2:1 (a weight ratio)) 10.0 kg, modified starch (made by MatsutaniChemical Industry Co, .Ltd, brand name: pine flow) 7.0 kg, tricalciumphosphate 0.5 kg, vitamin B₁ 0.4 kg, vitamin B₂ 0.4 kg, vitamin B₆ 0.5kg, and vitamin C 1.2 kg were tucked into blender and mixed for 10minutes. After the mixture was supplied into a tablet machine of thedirect compression formulation type and made a tablet (diameter of 7 mm,a height of 4 mm, 150 mg in weight), food in tablet form was producedexperimentally by coating with shellac thin-film using coating machine.This tablet can be used for purpose of enhancement of internal immunestrength, and prevention of lifestyle-related diseases such as diabetesmellitus or carcinoma.

Example 16

Butter 120 g, shortening 100 g, very-refined sugar 100 g and milk 100 mLwere put in bowl for families, and one egg was more added there whilewhipping together. After mixing enough, a mixture of sample 6, sample 7,and sample 8 (2:2:1, weight ratio) of the present invention 30 g wasadded there along with soft flour 200 g and baking powder 2 g, and thenthe mixture was kneaded enough together. After setting dough for 30minutes, the mixed compound was split into 50 using mould and burnt inoven, and then butter cookies were produced experimentally.

Example 17

Composition of the present invention comprising of sample 8, sample 10,and grape seed extract (made by Interhealthl company, brand name:Activin) (1:2:1 (a weight ratio)) 20 g were added to a commercialvegetable juice 1 L and mixed together, and then angiogenesis inhibitionvegetable juice was produced experimentally for use in antioxidation oftissue or prevention of diseases such as malignant, rheumatoidarthritis, and diabetes mellitus. There was no inferiority in this incomparison with original vegetable juice at all.

Example 18

A mixture of sample 6/sample 10=1/1 (a weight ratio) 100 kg, Ginkgobiloba extract 20 kg, shark cartilage extract 30 kg, yellow beeswax 10kg and corn oil 140 kg were mixed enough to give homogeneous liquidthing while warming to 80 degrees Celsius. This was supplied to thecapsule filling up machine, and then gelatine covered-capsuleformulation, of which quantity of one grain was 250 mg, was producedexperimentally. This formulation can be used as edible composition (foodand drink) or composition for medicine (drug) that oral ingestion ispossible.

This invention is not limited to the above embodiments and explanationthereof, and variations and modifications can be effected within thescope which does not depart from the description in the claims and canbe easily conceived by a person having ordinary skill in the art.

1. A method for inhibiting angiogenesis, in connection with treatingLewis Lung Cancer Cells or rheumatoid arthritis, which comprisesadministering to a person or an animal in need thereof a compositionselected from the group consisting of Formula (1) as an isolatedcompound or, a hydrochloride salt thereof, pyroglutamic acid, and sodiumsalt of pyroglutamic acid, including a pharmaceutically acceptablecarrier or an edible carrier


2. The method according to claim 1, wherein said compound of formula (1)and pyroglutamic acid are L-type or DL-type.